K562细胞DHRS4 L1基因新选择性剪接亚型的克隆及二级结构预测

Cloning of Novel Alternative Spliced Isoforms of DHRS4L1 Gene from K562 Cells and Prediction of Their Secondary Structure

目的： K562细胞DHRS4L1[ dehydrogenase／reductase （ SDR family） member 4 like 1]的表达及其二级结构分析。方法以K562细胞cDNA为模板， PCR扩增DHRS4[ dehydrogenase／reductase （ SDR fami-ly） member 4]基因簇Ea2转录本。将PCR产物A-T克隆至pGEMT-Easy质粒，对质粒进行DNA Sanger测序。将测序所得序列用NCBI ORF finder分析是否存在编码区，用Clustal Omega分析RNA序列系统树，用RNAfold web server分析RNA最小自由能二级结构。结果用RT-PCR和Sanger测序方法发现， K562表达DHRS4L1 Ea2转录本，未检测到其表达DHRS4L2[dehydrogenase／reductase （SDR family） member 4 like 2] Ea1转录本。 K562表达的DHRS4L1 Ea2至少有8种选择性剪接亚型（ KU058702、 KU058703、 KU058704、KU058705、 KU058706、 KU058707、 KU058708、 KU058709），其中6种为首次发现。 KU058702、 KU058703、KU058704、 KU058705和KU058707第二外显子受到多种形式的选择性剪接，恰好仅影响第2臂的二级结构，不影响其他臂的二级结构，提示这些不同亚型的二级结构有一定相似性，第二外显子所对应的二级结构臂可能在区分不同 DHRS4L1亚型功能中发挥关键作用。结论研究发现 K562细胞至少表达8种DHRS4L1选择性剪接亚型，为长链非编码RNA。其二级结构分析为后续研究DHRS4L1在白血病细胞中的潜在功能奠定基础。

Objective To examine the expression of dehydrogenase/reductase （ SDR family ） member 4 like 1 （ DHRS4L1） in K562 cells and its secondary structure.Methods Ea transcripts of DHRS4 gene cluster were amplified by PCR, with cDNA of K562 cells as the template.PCR products were cloned to pGEMT-Easy vector, which was followed by DNA Sanger sequencing and a-nalysis of whether sequences contains open reading frame using NCBI ORF finder.Phylogenetic tree analysis of RNA sequences was performed using Clustal Omega server.Analysis of the minimum free energy secondary structure of RNA was carried out using RNAfold web server.Results RT-PCR and Sanger sequencing showed that DHRS4L1 Ea2 transcripts were expressed in K562 cells, and＆amp;nbsp;DHRS4 L2 Ea1 expression was not detectable.At least 8 alternative spliced isoforms of DHRS4 L1 Ea2 （ KU058702, KU058703, KU058704, KU058705, KU058706, KU058707, KU058708, KU058709 ） were expressed in K562 cells, in which 6 of them were identified for the first time.The second exons of 5 transcripts （KU058702, KU058703, KU058704, KU058705 and KU058707） were subjected to extensive alternative splicing, which only affected the structure of the second arm of RNAs, but not other arms.This suggested that the secondary structure among these 5 different isoforms were similar.The second exon might have the key role in differentiating the functions of dif-ferent DHRS4L1 isoforms.Conclusion There are at 8 alternative spliced isoforms of DHRS4L1 and they are LncRNAs expressed in K562 cells.Analysis of DHRS4 L1 secondary structure would contrib-ute to the study of DHRS4 L1 potential functions in leukemia cells.